This invention relates to plasmin. More particularly, this invention relates to a synthetic substrate for the colorimetric or fluorometric assay of plasmin.
In man, fibrinolysis is controlled and regulated by the activity of the plasminogen-plasmin proteolytic enzyme system. Plasminogen, the naturally occurring precursor, is converted to plasmin by naturally occurring plasminogen activators or by kinases, such as streptokinase, urokinase, and staphylokinase. Plasminogen, normally present in all body fluids and secretions, has its highest concentration in plasma.
In the prior art, plasmin assays typically involved protein digestion systems, with casein being the most commonly used protein. Although such systems were satisfactory to a limited extent, a number of disadvantages are inherent in such systems. In general, protein digestion systems require at least about 15 minutes per assay; during this period of time, it is not possible to measure kinetic changes (e.g., the instantaneous formation of plasmin). Furthermore, it is not possible to evaluate the presence of plasminogen activators. Additionally, at high plasmin levels, turbid solutions often result. More importantly, protein digestion systems are not entirely reproducible from one source of protein to another (hence, from one laboratory to another). Protein digestion systems require the use of an ultraviolet spectrophotometer, which poses problems in determining the appropriate blank since digestion of serum proteins in the blank contributes to ultraviolet absorption. Furthermore, plasmin content cannot be defined in the traditional manner of micromoles of substrate hydrolyzed per unit of time. That is, the hydrolysis is not linear with enzymatic activity; an arbitrary curve is obtained which must be defined by a standard enzyme solution of limited availability.
Attempts to overcome the disadvantages of protein digestion systems in the assay of plasmin have led to the preparation of a number of synthetic substrates. Examples of such synthetic substrates include, among others, the following: ethyl p-guanidinobenzoate hydrochloride and p-nitrophenyl p'-guanidinobenzoate hydrochloride [T. Chase, Jr. and E. Shaw, Biochemistry, 8, 2212 (1969)]; N-(p-carboxybenzyl)pyridinium bromide p-nitrophenyl ester [J. M. Sodetz and F. J. Castellino, Biochemistry, 11, 3167 (1972)]; N.sup..alpha. -tosyl-L-arginine methyl, ethyl, and butyl esters, ethyl esters of glycine, L-lysine, DL-valine, L-leucine, L-isoleucine, DL-methionine, L-tyrosine, and DL-tryptophan, N.sup..alpha. -benzoyl-L-arginine ethyl ester, N.sup..alpha. -acetyl-L-tyrosine ethyl ester, N.sup..alpha. -acetyl-DL-tryptophan ethyl ester, and N.sup..alpha. -acetyl-DL-methionine ethyl ester [W. Troll, et al., J. Biol. Chem., 208, 85 (1954)]; L-arginine methyl ester, L-lysine methyl ester, N.sup..alpha. -acetyl-L-lysine methyl ester, N.sup..alpha. -benzoyl-L-arginine methyl ester, N.sup..alpha. -carbobenzoxy-L-lysine methyl ester, N.sup..alpha. -tosyl-L-lysine methyl ester, N.sup..alpha. -carbobenzoxy-L-arginine methyl ester, and N.sup..alpha. -acetyl-L-arginine methyl ester [S. Sherry, et al., Thromb. Diath. Haemorrh., 34, 20 (1975)]; N.sup..alpha. -benzyloxycarbonyl-L-lysine p-nitrophenyl ester [R. M. Silverstein, Thrombos. Res., 3, 729 (1973)]; N.sup..alpha. -methyl-N.sup..alpha. -tosyl-L-lysine .beta.-naphthol ester [P. H. Bell, et al., Anal. Biochem., 61, 200 (1974)]; and N.sup..alpha. -benzoylphenylalanine-valine-arginine-p-nitroanilide [P. Friberger, et al., Thromb. Diath. Haemorrh., 34, 321 (1975)]. The usual problems with most such synthetic substrates include, among others, a slow rate of reaction, rapid spontaneous hydrolysis, and difficulty in measuring the hydrolysis products.
It should be noted that some tripeptides are known which have the same amino acid sequences as the tripeptidyl portion of some of the substrates of the present invention. Examples of such known tripeptides include the following: N.sup..alpha. -trityl-glycine-glycine-N.sup..epsilon. -benzyloxycarbonyl-L-lysine benzyl ester, glycine-glycine-L-lysine, glycine-glycine-N.sup..epsilon. -benzyloxycarbonyl-L-lysine benzyl ester hydrochloride, and N.sup..alpha. -benzyloxycarbonyl-glycine-glycine-N.sup..epsilon. -benzyloxycarbonyl-L-lysine benzyl ester and hydrazide [O. Abe, et al., Bull. Chem. Soc. Japan, 40, 1945 (1967)]; N.sup..alpha. -formyl-L-phenylalanine-L-leucine-N.sup..epsilon. -t-butyloxycarbonyl-L-lysine and methyl ester thereof [L. V. Ionova and E. A. Morozova, J. Gen. Chem. USSR, 34, 407 (1964)]; N.sup..alpha. -benzyloxycarbonyl-glycine-glycine-L-lysine and diacetate monohydrate thereof [K. Suzuki and T. Abiko, Chem. Pharm. Bull. (Tokyo), 16, 1997 (1968)]; and O-benzyl-N.sup..alpha. -benzyloxycarbonyl-L-tyrosine-L-serine-N.sup..epsilon. -t-butyloxycarbonyl-L-lysine methyl ester, N.sup..alpha.,O-bis(benzyloxycarbonyl)-L-tyrosine-L-serine-N.sup..epsilon. -t-butyloxycarbonyl-L-lysine methyl ester, N.sup..alpha. -benzyloxycarbonyl-L-tyrosine-L-serine-N.sup..epsilon. -t-butyloxycarbonyl-L-lysine methyl ester, and L-tyrosine-L-serine-N.sup..epsilon. -t-butyloxycarbonyl-L-lysine methyl ester [A. A. Costopanagiotis, et al., J. Org. Chem., 33, 1261 (1968)].
The assay of plasmin by the plasmin-catalyzed hydrolysis of a given substrate to give one or more identifiable and measurable products is, of course, known in the art. The substrates of the present invention, however, can be used in such known procedure or procedures related thereto without the disadvantages attending the known, prior art substrates.